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LEGERMass SpectrometryBottom-up proteomicsbacillus cytotoxicusproteomicsmetabolomicsBacillus thuringiensisBacillus thuringiensis; bacillus cytotoxicus; proteomics; metabolomics; enterotoxins; caco-2enterotoxinscaco-2https://www.ebi.ac.uk/pride/archive/projects/PXD060436Caco-2 CellCell CultureEnterocyteBacterial supernatants Five strains of B. thuringiensis and one strain of B. cytotoxicus (Table 1) were amplified on Typton Soya Agar Yeast Extract (TSA-YA) medium (Biomérieux) for 18-24 hours at 30°C. Following this, isolated colonies were pre-cultured for 18 hours at 30°C in 10 mL of Brain Heart Infusion (BHI, Biomérieux). Subsequently, 10 mL of BHI medium were inoculated with a starting optical density at OD600nm=0.05 and incubated at 37°C, and 300 rpm for 5 hours, reaching an optical density ranging from 1.5 to 2. After centrifugation at 600 rpm for 20 minutes, and filtration through a 0.22µm filter to remove any remaining bacteria, the resulting cultures were aliquoted and stored at –20°C. Cell culture After thawing, 3.106 of the human Caco-2 cells (ATCC – HTB-37, Manassas, VA, USA), derived from colon adenocarcinoma, was seeded in a 75-cm2 flask and passaged for 12 to 34 times, when they reached approximately 80% confluence. The DMEM medium (Gibco – 41965-039) was supplemented with 10% fetal bovine serum (Gibco – 10270-106) as well as 1% non-essential amino acids (Gibco – 1114-035), 50 IU/mL of penicillin and 50 μg/mL of streptomycin (Gibco – 15140-122). Cells were seeded in 96-well microplates (20,000 cells/well) for assays on viability and pro-inflammatory effect, and in 12-well inserts (100,000 cells/insert) for omics experiments, and left to differentiate (up to 24-30 days). Cell treatments In the 96-well plates, the differentiated Caco-2 cells were treated for 24 hours by various dilutions of bacterial supernatants (from 0.1 to 12.5%) in DMEM medium described previously without FCS. For proteomic experiments, the Caco-2 cells in 12-well plates were treated for 24 hours with three selected non-cytotoxic dilutions of bacterial supernatants, determined from the results of the viability assay. Four independent experiments providing biological replicates for omics were carried out. For all the assays, BHI medium (Oxoid) was added to obtain a final concentration of 12.5% in all the wells, including the control ones. After the 24-hours treatment, the culture media were stored at -80°C for subsequent untargeted extracellular proteomic analyses. Proteomic experiments The equivalent of 500 µl was used for the extracellular proteomics sample preparation (derived from the medium, i.e., the secretome). After protein precipitation with acetone at -20°C overnight, as described previously, the samples were centrifuged for 5 minutes at 14,000 g and 4°C. The pellet was subsequently reconstituted in a 7 M urea solution and incubated at RT for 30 min. The proteins were reduced with 0.5 M TCEP for 30 min and alkylated with 0.5 M IAA for 20 min at RT. The sample was diluted with 25 mM ammonium bicarbonate. Proteins were digested by trypsin (Thermo Fisher Scientific) at a concentration of 200 ng/mL overnight at 37°C. Then, 1% TFA was added to the solution to stop the digestion. To purify the generated peptides, a solid-phase extraction (SPE) with Pierce C18 Tips (100µL bed, Thermo Fisher Scientific), prepacked columns filled with C18 resin, was performed. Eluates containing peptides were evaporated and the remaining peptides were reconstituted in 0.1% formic acid in HPLC-grade water before LC-MS/MS analysis. Proteomic analysis was additionally conducted on all the bacterial supernatants without any contact with the intestinal cells. To achieve this, 500 µl of each bacterial supernatant was processed, following the same sample preparation procedure as described above. Tandem mass spectrometry coupled to liquid chromatography The extracellular digested extracts were loaded into vials and injected into the Ultra High-Performance Liquid Chromatography (UHPLC) Vanquish (Thermo Fisher Scientific). Separation was achieved using an Acquity UPLC HSS T3 analytical column (1.8µm, 1mm x 150mm, 100 Å, Water), with two chromatographic mobile phases as described previously (39, 40): mobile phase A containing 0.1% AF in H20 and mobile phase B containing 0.1% AF in 100% ACN. The runs were performed at a flow rate of 80 µL/min. A gradient elution was applied as follows: starting from 95% of mobile phase A, the percentage of mobile phase B was increased to 40% over 98 minutes. Subsequently, a 23 minutes column regeneration step was performed, resulting in a total elution time of 2 hours. The UHPLC system was coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). To generate fragments, HCD activation was used with a collision energy of 27% and an isolation width of 1.4 Da. MS/MS data were acquired using a data-dependent acquisition mode, where the 20 most intense precursor ions were selected for fragmentation. A dynamic exclusion time of 30 seconds and a resolution of 17,500 were implemented. The automatic gain control was set to 3e6 for MS and 2e5 for MS/MS, respectively. Maximum ion accumulation times of 50 ms for MS and 45 ms for MS/MS were established.PrideProteomic data analysis Proteomic data were analyzed using MaxQuant version 2.0.3.1 and searched with the Andromeda search engine against the UniProt Homo sapiens database (Version 2021) as previously described (39) which included entries for the major toxins of Bt (158), Bcyt (27), and Bc (1). A mass deviation of 20 ppm was set for the parent mass (MS1), and 4.5 ppm for the fragment ions (MS2). The minimum peptide length was 5 amino acids, and strict trypsin cleavage specificity was applied, allowing up to two missed cleavage sites. Carbamidomethylation (Cys) was set as a fixed modification, and oxidation of methionine (Met), acetylation (N-Terminal), phosphorylation (STY), as well as ubiquitination of lysine (K) were considered as variable modifications. False discovery rates (FDRs) at both the protein and peptide levels were set to 1%. The option "match between runs" (MBR) was activated to improve peptide identification and quantification across different samples. Peptides and proteins abundance variation were measured using the LFQ quantification algorithm. The statistical analyses for the proteomic data were performed using Perseus software version 1.6.15 as described previously (39). Total proteins were taken into account if they had one valid abundance values. A normal distribution with a width of 0.3 and a downshift parameter of 1.8 was used to impute missing values. Protein abundance differences were considered significant if the corresponding estimated ANOVA p-values were less than 0.05. A Student's t-test was used for each comparison of two experimental conditions. The permutation-based FDR threshold and the number of randomizations were both set to 0.05 and 250, respectively. ClueGO, a Cytoscape plug-in version 3.8.2, was used to generate significant GO terms enriched with an adjusted p-value < 0.05 (Benjamini-Hochberg correction), using all quantified proteins as the reference set. These significant GO terms were then visualized using an R script specifically designed for GO term visualization, protein interaction network were obtained from STRING version 12.0. All other figures, including volcano plots and boxplots, were generated using R version 4.3.1.ProteomicsThibaut LégerAnses FougèresCOMPLETEBacillus CytotoxicusHomo Sapiens (human)Bacillus ThuringiensisBacteriathibaut.leger@anses.fr41932230 Alilat S, Lanceleur R, Bonis M, Pairaud S, De Almeida NP, Vial G, Sarda X, Firmesse O, Fessard V, Liuu S, Léger T. Multi-omics characterisation of the virulence and inflammatory potential of Bacillus thuringiensis in an in vitro intestinal epithelial model. Sci Total Environ. 2026 1029:181742 10.1016/j.scitotenv.2026.181742French Agency for Food, Environmental and Occupational Health & Safety (ANSES)France10.6019/PXD060436The soil bacterium Bacillus thuringiensis (Bt) can produce spores and crystals composed of toxins with insecticidal activity. Due to these properties, some Bt strains have been developed as biopesticides for pest control, offering an alternative to chemical insecticides. However, Bt strains belong to the Bacillus cereus (Bc) group and share some virulence genes encoding diarrhoeal enterotoxins such as Hemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe) and Cytotoxin K (Cyt-K2). These toxins are known to be involved in foodborne outbreaks (FBOs) caused by Bc. For this reason, it has been suggested that Bt may also cause FBOs, making it crucial to evaluate their virulence potential and effects on human health. To address this, differentiated human intestinal Caco-2 cells, which mimic the intestinal barrier, were treated with serial dilutions of culture supernatants from five Bt strains used as biopesticides. No cytotoxicity was observed with any of the Bt supernatants after 24 h treatment in contrast to Bacillus cytotoxicus (Bcyt) supernatants. However, interleukin 8 (IL-8) secretion increased sharply for Bt as well as Bcyt, suggesting a potential for chronic inflammation. To investigate the molecular effects of Bt supernatants on Caco-2 cells, untargeted proteomics and metabolomics were performed on cell media and lysates using two non-cytotoxic concentrations. Both approaches highlighted common perturbations on oxidative phosphorylation and lipid metabolism for exposure to Bt or Bcyt supernatants. Proteins linked to acute inflammation decreased only with Bcyt exposure. Bt supernatants, but not Bcyt supernatants caused shedding of CD59 and CD55, two membrane-bound complement inhibitors, into the culture medium. This demonstrated that Bt supernatants could increase the vulnerability of intestinal cells to their own immune system. Proteomics also revealed the heterogeneous toxin abundances in the supernatants of the different Bt subspecies, highlighting the need to assess each strain individually when assessing the risks associated with their use as biopesticides.Multi-omics characterisation of the virulence and inflammatory potential of Bacillus thuringiensis in an in vitro intestinal epithelial model.Alilat Sarah S, Lanceleur Rachelle R, Bonis Mathilde M, Pairaud Sylvie S, De Almeida Natália Pinto NP, Vial Gaëlle G, Sarda Xavier X, Firmesse Olivier O, Fessard Valérie V, Liuu Sophie S, Léger Thibaut TfalseProteomic analysis of raw bacillus thuringiensis supernatants and those used to expose Caco-2 cellsThe soil bacterium Bacillus thuringiensis (Bt) can produce spores and crystals composed of toxins with insecticidal activity. Due to these properties, some Bt strains have been developed as biopesticides for pest control, with a high value to restrict the use of chemical insecticides. However, Bt strains belong to the Bacillus cereus (Bc) group, sharing some virulence genes encoding diarrheal enterotoxins like hemolysin BL (Hbl), non-hemolytic enterotoxin (Nhe) and cytotoxin K (Cyt-K2). These toxins are known to be involved in foodborne outbreaks (FBOs) caused by Bc sensu stricto contamination. For this reason, it has been suggested that Bt may also provoke FBOs and it is therefore crucial to evaluate their virulence potential and to characterize their effects on humans. For this purpose, differentiated human intestinal Caco-2 cells, able to mimic the human intestinal barrier, have been treated with serial dilutions of the culture supernatant of five different Bt strains used as biopesticides. No cytotoxicity was induced with all Bt supernatants after 24 h treatment in contrast to Bacillus cytotoxicus (Bcyt) supernatants. Interleukin 8 (IL-8) secretion increased sharply in both groups, suggesting possible concerns with chronic inflammation. To investigate the molecular effects of Bt supernatants on Caco-2 cells, untargeted proteomics and metabolomics were performed on both cell media and lysates with 3 non-cytotoxic concentrations. Proteomics and metabolomics highlighted common perturbations on oxidative phosphorylation (OXPHOS) and lipid metabolism for both Bt and Bcyt supernatants exposures. Proteins linked to acute inflammation decreased only with Bcyt exposure. Bt supernatants and not Bcyt supernatants caused a shedding of CD59 and CD55, inhibitors of the complement system, from the cell membrane into the culture medium. This showed that Bt supernatants can increase the vulnerability of intestinal cells to their own immune system. Proteomics also revealed the heterogeneity of toxin abundance in the supernatants of the different Bt subspecies, demonstrating the importance of taking subspecies into account when assessing the risks associated with their use as 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